1978 National Cancer Institute CARCINOGENESIS Technical Report Series No. 30 BIOASSAYOF DIARYLANILIDE YELLOW FOR POSSIBLE CARCINOGENICITY CAS No. 6358-85-6 NCI-CG-TR-30 U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE Public Health Service National Institutes of Health
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1978
National Cancer Institute
CARCINOGENESIS Technical Report Series No. 30
BIOASSAYOF
DIARYLANILIDE YELLOW
FOR POSSIBLE CARCINOGENICITY
CAS No. 6358-85-6
NCI-CG-TR-30
U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE Public Health Service National Institutes of Health
BIDASSAY OF
DIARYLANILIDE YELLOW
FOR POSSIBLE CARCINOGENICITY
Carcinogenesis Testing Program Division of Cancer Cause and Prevention
National Cancer Institute National Institutes of Health
Bethesda, Maryland 20014
U.S. DEPARTMENT OF HEALTH, EDUCATION, AND WELFARE Public Health Service
National Institutes of Health
DHEW Publication No. (NIH) 78-830
REPORT ON THE BIOASSAY OF DIARYLANILIDE YELLOW FOR POSSIBLE CARCINOGEN1CITY
CARCINOGENSSIS PROGRAM, DIVISION OF CANCER CAUSE AND PREVENTION NATIONAL CANCER INSTITUTE, NATIONAL INSTITUTES OF HEALTH
CONTRIBUTORS: This report presents the results of the bioassay of diarylanilide yellow conducted for the Carcinogen Bioassay and Program Resources Branch, Carcinogenesis Program, Division of Cancer Cause and Prevention, National Cancer Institute (NCI), National Institutes of Health, Bethesda, Maryland. This bioassay was conducted by Mason Research Institute, Worcester, Massachusetts, initially under direct contract to the NCI and currently under a subcontract to Tracer Jitco, Inc. (1), prime contractor for the NCI Carcinogenesis Bioassay Program.
The experimental design was determined by the NCI Project Officers, Dr. J. H. Weisburger (2,3) and Dr. E. K. Weisburger (2). The principal investigators for the contract were Dr. E. Smith (4) and Dr. A. Handler (4). Animal treatment and observation were supervised by Mr. G. Wade (4) and Ms. E. Zepp (4). Chemical analysis was performed by Midwest Research Institute (5).
Histopathology was performed by Dr. R. W. Fleischman (4) at the Mason Research Institute, and the diagnoses included in this report represent the interpretation of this pathologist.
Compilation of individual animal survival, pathology, and summary tables was performed by EG&G Mason Research Institute (6); the statistical analysis was performed by Dr. A. Chu (6) and Mr. W. W. Belew (7), using methods selected for the Bioassay Program by Dr. J. J. Gart (8).
This report was prepared at METREK, a Division of The MITRE Corporation (7) under the direction of the NCI. Those responsible for this report at METREK are the project coordinator, Dr. L. W. Thomas (7), the task leader, Dr. M. R. Kornreich (7), and the senior biologist, Ms. P. Walker (7). The final report was reviewed by members of the participating organizations.
The statistical analysis was reviewed by a member or members of the Mathematical Statistics and A plied Mathematics Section of the NCI: Dr. J. J. Gart (8), Mr. J. i.am (8), Dr. H. M. Pettigrew (8), and Dr. R. E. Tarone (8).
iii
The following other scientists at the National Cancer Institute were responsible for evaluating the bioassay experiment, interpreting the results, and reporting the findings: Dr. K. C. Chu (2), Dr. C. Cueto, Jr. (2), Dr. J. F. Douglas (2), Dr. D. G. Goodman (2), Dr. R. A. Griesemer (2), Di. T. W. Orme (2), Dr. R. A. Squire (9), and Dr. J. M. Ward (2).
1. Tracor Jitco, Inc., 1776 East Jefferson Street, Rockville, Maryland.
2. Carcinogenesis Program, Division of Cancer Cause and Prevention, National Cancer Institute, National Institutes of Health, Bethesda, Maryland,
3. Now with the Nayloi: Dana Institute for Disease Prevention, American Health Foundation, Hammon House Road, Valhalla, New York.
4. Mason Research Institute, 57 Union Street, Worcester, Massachusetts.
5. Midwest Research Institute, 425 Volker Boulevard, Kansas City, Montana.
6. EG&G Mason Research Institute, 1530 East Jefferson Street, Rockville, Maryland.
7. The MITRE Corporation, METREK Division, 1820 Dolley Madison Boulevard, McLean, Virginia.
8. Mathematical Statistics and Applied Mathematics Section, Biometry Branch, Field Studies and Statistics Program, Division of Cancer Cause and Prevention, National Cancer Institute, National Institutes of Health, Buthesda, Maryland.
9. Now with the Division of Comparative Medicine, Johns Hopkins University, School of Medicine, Traylor Building, Baltimore, Maryland.
iv
SUMMARY
A bioassay of technical-grade diarylanilide yellow for possible carcinogenicity was conducted using Fischer 344 rats and B6C3F1 mice. Diarylanilide yellow was administered in the feed, at either of two concentrations, to groups of 50 male and 50 female animals of each species. The high and low dietary concentrations used in the chronic study for the male and female rats and mice were 5.0 and 2.5 percent, respectively, of the chemical in the feed. After a 78-week treatment period, observation of the rats continued for an additional 28 weeks and observation of the mice continued for an additional 19 weeks for high dose males and low and high dose females and 18 weeks for low dose males. For each species, 50 animals of each sex were placed on test as controls, and fed only the basal diet.
The high concentration administered to both species in this study was the maximum recommended in the Guidelines for Carcinogen Bioassay in Small Rodents (Sontag et al., 1976). These guidelines indicate that a chronic dietary level of 5 percent, or 50,000 ppm, should not be exceeded even when no signs of toxicity are observed during sub-chronic testing, except under special circumstances (e.g., when the compound is a major component of the human diet). No toxic effects were reported during subchronic testing and diarylanilide yellow did not qualify for exception; therefore, the highest permissible concentration (5 percent) was utilized in the chronic bioassay.
The dietary concentrations of diarylanilide yellow administered during the chronic bioassay had no significant effect on survival or body weight gain in either species. Except for yellow staining and some isolated neoplasms, the only adverse clinical sign or pathologic lesion observed in treated rats or mice was basophilic cytoplasm changes in hepatocytes of treated rats.
In both species the survival in all groups was adequate for statistical analysis of late-appearing tumors.
No treatment-related increase in the incidence of neoplasms or nonneoplastic lesions was evident in treated rats or mice. A few unusual findings were observed in both species, including single cases of metastatic chordoma and ost.eogenic sarcoma in rats, and single cases of squamous-cell carcinoma of the ear, infiltrating duct carcinoma of the mammary gland, and subcutaneous mastocytoma in mice.
The results of this study did not provide evidence for the carcinogenicity of diarylanilide yellow in Fischer 344 rats or B6C3F1 mice.
v
TABLE OF CONTENTS
I. INTRODUCTION
II. MATERIALS AND METHODS
A. Chemicals B. Dietary Preparation C. Animals D. Animal Maintenance E. Selection of Initial Concentrations F. Experimental Design G. Clinical and Histopathologic Examinations H. Data Recording and Statistical Analyses
III. CHRONIC TESTING RESULTS: RATS
A. Body Weights and Clinical Observations B. Survival C. Pathology D. Statistical Analyses of Results
IV. CHRONIC TESTING RESULTS: MICE
A. Body Weights and Clinical Observations B. Survival C. Pathology D. Statistical Analyses of Results
V. DISCUSSION
VI. BIBLIOGRAPHY
APPENDIX A SUMMARY OF THE INCIDENCE OF NEOPLASMS IN RATS TREATED WITH DIARYLANILIDE YELLOW
APPENDIX B SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MICE TREATED WITH DIARYLANILIDE YELLOW
APPENDIX C SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN RATS TREATED WITH DIARYLANILIDE YELLOW
APPENDIX D SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MICE TREATED WITH DIARYLANILIDE YELLOW
Page
1
3
3 3 A 4 6 8 8 12
17
17 17 20 20
26
26 26 29 30
33
35
A-l
B-l
C-l
D-l
vii
Figure Number
1
2
3
4
Table Number
1
2
3
4
5
6
Al
LIST OF ILLUSTRATIONS
GROWTH CURVES FOR DIARYLANILIDE YELLOW CHRONIC STUDY RATS
SURVIVAL COMPARISONS OF DIARYLANILIDE YELLOts CHRONIC STUDY RATS
GROWTb CURVES FOR DIARYLANILIDE YELLOW CHRONIC STUDY MICE
SURVIVAL COMPARISONS OF DIARYLANILIDE YELLOW CHRONIC STUDY MICE
LIST OF TABLES
DESIGN SUMMARY FOR FISCHER 344 RATS— DIARYL\NILIDE YELLOW FEEDING EXPERIMENT
DESIGN SUMMARY FOR B6C3F1 MICE—DIARYLANILIDS YELLOW FEEDING EXPERIMENT
ANALYSIS OF THE INCIDENCE OF PRIMARY TUMORS AT SPECIFIC SITES IN MALE RATS TREATED WITH DIARYLANILIDE YELLOW
ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT SPECIFIC SITES IN FEMALE RATS TREATED WITH DIARYLANILIDE YELLOW
ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT SPECIFIC SITES IN MALE MICE TREATED WITH DIARYLANILIDE YELLOW
ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT SPECIFIC SITES IN FEMALE MICE TREATED WITH DIARYLANILIDE YELLOW
SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE RATS TREATED WITH DIARYLANILIDE YELLOW
Page
18
19
27
28
Page
9
10
21
23
31
32
A-3
viii
LIST OF TABLES (Concluded)
Table Number
A2 SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE RATS TREATED WITH DIARYLANILIDE YELLOW A ~ 7
Bl SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE MICE TREATED WITH DIARYLANILIDE YELLOW B ~ 3
B2 SUMMARY OF THE INCIDENCE OF NEOPLASMS IN FEMALE MICE TREATED WITH DIARYLANILIDE YELLOW B-6
Cl SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE RATS TREATED WITH DIARYLANILIDE YELLOW C-3
C2 SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE RATS TREATED WITH DIARYLANILIDE YELLOW c ~ 8
Dl SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE MICE TREATED WITH DIARYLANILIDE YELLOW D ~ 3
D2 SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN FEMALE MICE TREATED WITH DIARYLANILIDE YELLOW D ~ 7
I. INTRODUCTION
Diarylanilide yellow (NCI No. C03269), one member of a family of
organic azo pigments known as benzidine yellows, was selected for
bioassay by the National Cancer Institute in an attempt to elucidate
those dyes and dye intermediates which may be responsible for the
increased incidence of bladder cancer observed among workers in the
dye manufacturing industry (Wynder et al., 1963; Anthony et al.,
1970). The structural relationship of this compound to the documented
carcinogen 3,3'-dichlorobenzidine (Occupational Safety and Health
Administration, 1973) was also a factor in its selection.
The Chemical Abstracts Service (CAS) Ninth Collective Index
(1977) name for this compound is 2,2'-[(3,3'-dichloroU,1'-biphenyl)
* 4,4'-diyl)-bis (azo)] bis (3-oxo-N-phenyl)-butanamide. It is also
called Color Index (C.I.) Pigment Yellow 12 (C.I. 21090), diarylide
yellow, and dichlorobenzidine coupled into acetoacetanilide. Diaryl
anilide yellow is an ingredient in industrial paints, most notably
the paint applied to lead pencils (Weisburger, 1976). It is also an
ingredient in printing inks and may sometimes be used to color
plastics, rubber, linoleum, floor tiles, textiles, and wallpaper
(Society of Dyers and Colourists, 1971; Hawley, 1971). According to
the U.S. International Trade Commission (1977a), 6.028 x 10 pounds
* The CAS registry number is 6358-85-6.
1
of diarylani1ide yellow were produced in the United States in 1975—
the largest quantity of any single pigment produced in that year.
U.S. imports of the pigment through principle U.S. customs districts
amounted to 62,040 pounds in 1975 (U.S. International Trade Commis
sion, 1977b).
The risk of exposure to diarylanilide yellow is greatest among
workers in the dye marufacturing industry and at facilities where
dyeing of textiles or production of inks, paints, and other commodi
ties containing the pigment takes place. Additional occupational
exposure may also occur among users of pigment-containing products
(e.c., among printers engravers, lithographers, textile workers,
etc.) .
Epiaemioiogical studies suggest a relationship between occupa
tional exposure to pa .nts and increased incidences of cancer of the
lung and bladder and between occupational exposure to printing inks
and increased incidences of cancer of the liver and bladder (Hoover
and Fraumeni, 1975). An increased incidence of bladder cancer has
also been observed among textile workers and tailors (Anthony and
Thomas, 1970).
Exposure of the general population to diarylanilide yellow is
likely, due to the large variety of consumer products colored with
this pigment. Chroni: ingestion of the dye over long periods of time
may result from habitjal holding in the mouth or chewing of wooden
pencils.
2
II. MATERIALS AND METHODS
A. Chemicals
Diarylanilide yellow was purchased from Chemtron Corporation and
chemical analysis was performed by Midwest Research Institute. The
melting point range (311° to 320°C) suggested the presence of impuri
ties. Thin-layer chromatography was performed utilizing two different
solvent systems (methylene chloride and 95:5 chloroform:diethylamine).
Each plate was visualized with ultraviolet and visible light. One
homogeneous spot was detected on each plate; however, the amounts of
compound spotted on each plate (2.4 |ag and 7.2 |j.g) were so low that
only major impurities could have been detected by this technique.
Infrared and mass spectrometry analyses were not inconsistent with
the structure of the compound.
Throughout this report the term diarylanilide yellow is used to
represent this technical-grade material.
B. Dietary Preparation
The basal laboratory diet for both treated and control animals
(F consisted of Wayne Lab-Blox (Allied Mills, Inc.). Diarylanilide
yellow was administered to the treated animals as a component of the
diet. The chemical was mixed in the feed in a 6 kg capacity
Patterson-Kelly standard model stainless steel twin-shell V-blender.
After 20 minutes of blending, the mixtures were placed in double
plastic bags and stored in the dark at 4°C. Mixtures were prepared
weekly and stored for not longer than 2 weeks.
3
C. Animals
Two animal species, rats and mice, were used in the carcinogeni
city bioassay. The Fischer 344 rats and the B6C3F1 mice were obtained
through contracts of the Division of Cancer Treatment, National Cancer
Institute. Animals of both species were supplied by Charles River
Tissues for which slides were prepared were preserved in 10 per
cent buffered formalin, embedded in paraffin, sectioned, and stained
with hematoxylin and eosin prior to microscopic examination. An
occasional section was subjected to special staining techniques for
more definitive diagnosis.
A few tissues were not examined for some animals, particularly
for those that died early. Also, some animals were missing, canni
balized, or judged to be in such an advanced state of autolysis as to
11
preclude histopathologLc interpretation. Thus, the number of animals
for which particular organs, tissues, or lesions were examined micro
scopically varies and does not necessarily represent the number of
animals that were placed on experiment in each group.
H. Data Recording and Statistical Analyses
Pertinent data on this experiment have been recorded in an auto
matic data processing system, the Carcinogenesis Bioassay Data System
(Linhart et al., 1974),, The data elements include descriptive infor
mation on the chemicals, animals, experimental design, clinical obser
vations, survival, body weight, and individual pathologic results, as
recommended by the International Union Against Cancer (Berenblum,
1969). Data tables were generated for verification of data transcrip
tion and for statistical review.
These data were analyzed using the statistical techniques
described in this section. Those analyses of the experimental results
that bear on the possibility of carcinogenicity are discussed in the
statistical narrative sections.
Probabilities of survival were estimated by the product-limit
procedure of Kaplan and Meier (1958) and are presented in this report
in the form of graphs. Animals were statistically censored as of the
time that they died of other than natural causes or were found to be
missing; animals dying from natural causes were not statistically
censored. Statistical analyses for a possible dose-related effect on
survival used the method of Cox (1972) for testing two groups for
12
equality and used Tarone's (1975) extensions of Cox's methods for
testing a dose-related trend. One-tailed P-values have been reported
for all tests except the departure from linearity test, which is only
reported when its two-tailed P-value is less than 0.05.
The incidence of neoplastic or nonneoplastic lesions has been
given as the ratio of the number of animals bearing such lesions at a
specific anatomic site (numerator) to the number of animals in which
that site was examined (denominator). In most instances, the denomi
nators included only those animals for which that site was examined
histologically. However, when macroscopic examination was required
to detect lesions prior to histologic sampling (e.g., skin or mammary
tumors), or when lesions could have appeared at multiple sites (e.g.,
lymphomas), the denominators consist of the numbers of animals necrop
sied.
The purpose of the statistical analyses of tumor incidence is to
determine whether animals receiving the test chemical developed a sig
nificantly higher proportion of tumors than did the control animals.
As a part of these analyses, the one-tailed Fisher exact test (Cox,
1970, pp. 48-52) was used to compare the tumor incidence of a control
group to that of a group of treated animals at each dose level. When
results for a number of treated groups, k, are compared simultaneously
with those for a control group, a correction to ensure an overall
significance level of 0.05 may be made. The Bonferroni inequality
(Miller, 1966, pp. 6-10) requires that the P-value for any comparison
13
be less than or equal t:> 0.05/k. In cases where this correction was
used, it is discussed i:i the narrative section. It is not, however,
presented in the tables, where the Fisher exact P-values are shown.
The Cochran-Armita^e test for linear trend in proportions, with
continuity correction (Armitage, 1971, pp. 362-365), was also used.
Under the assumption of a linear trend, this test determined if the
slope of the dose-response curve is different from zero at the one-
tailed 0.05 level of significance. Unless otherwise noted, the di
rection of the significant trend was a positive dose relationship.
This method also provides a two-tailed test of departure from linear
trend.
A time-adjusted analysis was applied when numerous early deaths
resulted from causes that were not associated with the formation of
tumors. In this analysis, deaths that occurred before the first tu
mor was observed were excluded by basing the statistical tests on
animals that survived at: least 52 weeks, unless a tumor was found at
the anatomic site of interest before week 52. When such an early
tumor was found, comparisons were based exclusively on animals that
survived at least as long as the animal in which the first tumor was
found. Once this reduced set of data was obtained, the standard pro
cedures for analyses of the incidence of tumors (Fisher exact tests,
Cochran-Armitage tests, etc.) were followed.
When appropriate, life-table methods were used to analyze the
incidence of tumors. Curves of the proportions surviving without an
14
observed tumor were computed as in Saffiotti et al. (1972). The week
during which animals died naturally or were sacrificed was entered as
the time point of tumor observation. Cox's methods of comparing
these curves were used for two groups; Tarone's extension to testing
for linear trend was used for three groups. The statistical tests for
the incidence of tumors which used life-table methods were one-tailed
and, unless otherwise noted, in the direction of a positive dose
relationship. Significant departures from linearity (P < 0.05, two-
tailed test) were also noted.
The approximate 95 percent confidence interval for the relative
risk of each dosed group compared to its control was calculated from
the exact interval on the odds ratio (Gart, 1971). The relative risk
is defined as p /p where p is the true binomial probability of the
incidence of a specific type of tumor in a treated group of animals
and p is the true probability of the spontaneous incidence of the
same type of tumor in a control group. The hypothesis of equality
between the true proportion of a specific tumor in a treated group
and the proportion in a control group corresponds to a relative risk
of unity. Values in excess of unity represent the condition of a
larger proportion in the treated group than in the control.
The lower and upper limits of the confidence interval of the
relative risk have been included in the tables of statistical analy
ses. The interpretation of the limits is that in approximately 95
percent of a large number of identical experiments, the true ratio
15
of the risk in a treated group of animals to that in a control group
would be within the interval calculated from the experiment. When
the lower limit of the confidence interval is greater than one, it
can be inferred that a statistically significant result (a P < 0.025
one-tailed test when the control incidence is not zero, P < 0.050
when the control incidence is zero) has occurred. When the lower
limit is less than unity but the upper limit is greater than unity,
the lower limit indicates the absence of a significant result while
the upper limit indicates that there is a theoretical possibility
of the induction of turners by the test chemical which could not be
detected under the conditions of this test.
16
III. CHRONIC TESTING RESULTS: RATS
A. Body Weights and Clinical Observations
The body weight patterns for control and treated rat groups of
both sexes were generally equivalent throughout the treatment period
(Figure 1).
All the treated rats, both male and female, appeared bright
yellow in color. In addition, the conjunctivas were faintly yellow
as were most organs and internal mucosal surfaces. The only other
clinical sign recorded for male or female rats was a hard crusted
lesion on the back of one male control animal.
B. Survival
The estimated probabilities of survival for male arid female rats
in the control and diarylanilide yellow-treated groups are shown in
Figure 2.
For both male and female rats the Tarone test detected no sta
tistically significant positive association between dosage and mor
tality. In the males survival was quite high, as 74 percent of the
high dose, 84 percent of the low dose, and 64 percent of the control
rats survived until the end of the study, despite the sacrifice of
five high dose and five control rats in week 78. In the females, 66
percent of the high dose, 80 percent of the low dose, and 72 percent
of the control rats survived until the end of the study, despite the
sacrifice of five high dose and five control rats in week 78.
17
TIME ON TEST (WEEKS)
FIGURE 1 GROWTH CURVES FOR DIARYLANILIDE YELLOW CHRONIC STUDY RATS
18
TIME ON TEST (WEEKS)
FIGURES SURVIVAL COMPARISONS OF DIARYLANILIDE YELLOW CHRONIC STUDY RATS
19
In both sexes, survival was adequate for meaningful statistical
analyses of tumor incidence.
C. Pathology
Histopathologic findings on neoplasms in rats are tabulated in
Appendix A (Tables Al and A2); findings on nonneoplastic lesions are
tabulated in Appendix C (Tables Cl and C2).
With a few exceptions, the same variety of neoplasms occurred
sporadically and randomly in the chemically treated and control
groups. No particular organ or system seemed to be the target of
this chemical. Sporadic and unusual neoplasms that occurred in the
treated but not in control animals were as follows: a metastatic
chordoma of unknown origin occurred in the lung of 1/49 of the low
dose males, and 1/49 of the low dose females had an osteogenic
sarcoma.
The incidence and variety of nonneoplastic degenerative, prolif
erative, and inflammatory lesions were similar in the control and
the chemically treated rats, except for treatment-related basophilic
cytoplasm changes in hepatocytes of treated males and females.
The results of this histopathologic examination did not provide
evidence for the carcinogenicity of diarylanilide yellow in Fischer
344 rats.
D. Statistical Analyses of Results
The results of the statistical analyses of tumor incidence in
rats are summarized in Tables 3 and 4. The analysis for every type
20
TABLE 3
ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT SPECIFIC SITES IN MALE RATS TREATED WITH DIARYLANILIDE YELLOW3
I 21
TOPOGRAPHY :MORPHOLOGY
Skin: Fibroma or Basal-cell Carcinoma
P Values0 >d
Relative Risk (Control)6
Lover Limit Upper Limit
Weeks to First Observed Turoor
Hematopoletic System: Leukemia or Malignant Lymphotna
P Values0 >d
Relative Risk (Control)6
Lower Limit Upper Limit
Weeks to First Observed Tumor
Pituitary: Adenoma
P Values0 'd
Departure from Linear Trend
Relative Risk (Control)6
Lower Limit Upper Limit
Weeks to First Observed Tumor
Adrenal: Pheochromocytoma
P Values0 >d
Relative Risk (Control)6
Lover Limit Upper Limit
Weeks to First Observed Tumor
LOW HIGH CONTROL DOSE DOSE
2/50(0.04) 4/50(0.08) 1/50(0.02)
N.S. N.S. N.S.
2.000 0.500 0.301 0.009 21.320 9.290
85 91 106
10/50(0.20) 2/50(0.04) 1/50(0.02)
P O.OOl(N) P 0.014(N) P 0.004(N)
0.200 0.100 0.022 0.002 0.877 0.662
78 99 103
7/45(0.16) 12/43(0.28) 5/45(0.11)
N.S. N.S. N.S.
P 0.041
1.794 0.714 0.723 0.193 4.856 2.414
78 106 106
3/50(0.06) 3/47(0.06) 5/49(0.10)
N.S. N.S. N.S.
1.064 1.701 0.149 0.351 7.570 10.420
78 96 106
TOPOGRAPHY: MORPHOLOGY
Thyroid: C-Cell Adenoma or Carcinoma
P Values0 >d
Relative Risk (Control)6
Lower Limit Upper Limit
Pancreatic Islets: Adenoma
P Values0 >d
Relative Risk (Control)6
Lower Limit Upper Limit
Weeks to First Observed Tumor
Test Is: Interstitial-Cell Tumor
P Values0 >d
Relative Risk (Control)6
Lower Limit Upper Limit
Weeks to First Observed Tumor
TABLE 3 (CONCLUDED)
LOU HIGH CONTROL DOSE DOSE
3/37(0.08) 5/47(0.11) 1/48(0.02)
N.S. N.S. N.S.
1.312 0.257 0.275 0.005 7.994 3.055
105 96 ICC
1/47(0.02) 2/47(0.04) 5/46(0.11)
N.S. N.S. JI.S.
2.000 5.109 0.108 0.603
115.500 235.900
109 106 93
42/50(0.84) 44/48(0.92) 39/49(0.80)
N.S. N.S. N.S.
1.091 0.948 0.922 0.782 1.240 1.161
78 96 78
22
Dosed groups received concentrations of 2.5 and 5.0 percent in feed.
Number of tumor-bearing animals/number of animals examined at site (proportion). CBeneath the incidence of the control is the probability level for the Cochran-Armitage test for dose-related trend in proportions when it Is below 0.05; otherwise N.S.- not significant. Departure from linear trend is noted when it is below 0.05 for any comparison. Beneath each dose group incidence is the probability level for the Fisher exact (conditional) test for the comparison of that dose group to the control group when it la below 0.05, otherwise N.S.- not significant.
A negative trend (N) indicates a lower incidence in a treated group than in a control group.
eRelative risk of the treated group versus the control group is shown along with the lower and upper limit of the 95% confidence interval for that relative risk.
8
TABLE 4
ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT SPECIFIC SITES IN FEMALE RATS TREATED WITH DIARYLANILIDE YELLOW8
T>osed groups received concentrations of 2.5 and 5.0 percent in feed.
Number of tumor-bearing animals/number of animals examined at site (proportion).
Beneath the incidence of the control is the probability level for the Cochran-Armltage test for dose-related trend in proportions when it is below 0.05; otherwise N.S. - not significant. Departure from linear trend is noted when it is below 0.05 for any comparison. Beneath each dose group Incidence is the probability level for the Fisher exact (conditional) test for the comparison of that dose group to the control group when it is below 0.05, otherwise N.S. - not significant.
A negative trend (N) indicates a lower incidence in a treated group than in the control group.
ilelatlve risk of the treated group versus the control group is shown along with the lower and upper limit of the 95Z confidence interval for that relative risk.
of tumor that was observed in more than 5 percent of any of the
diarylanilide yellow-dosed groups of either sex is included.
None of the statistical tests for rats of either sex indicated a
significant positive association between dosage and tumor incidence.
To provide additional insight, 95 percent confidence intervals
on the relative risk have been estimated and entered in the tables
based upon the observed tumor incidence rates. In many of the inter
vals shown in Tables 3 and 4, the value one is included; this indi
cates the absence of statistically significant results. It should
also be noted that many of the confidence intervals have an upper
limit greater than one, indicating the theoretical possibility of a
significantly increased rate of tumor incidence induced in rats by
diarylanilide yellow that could not be established under the condi
tions of this test.
25
IV. CHRONIC TESTING RESULTS: MICE
A. Body Weights and Clinical Observations
No d i f fe rences between body weight gain patterns of high dose
groups and low dose groups were evident in male or female mice during
the 78-week treatment period (Figure 3). The control animals for
both sexes began to experience marked weight gain beginning in week
36 when compared to ths treated mice.
All the treated mice, both male and female, acquired a yellow
discoloration of the hair coat during treatment. Because of the
normal darker color of the B6C3F1 mice, the external appearance of
the mice was not as strikingly af fec ted as that of the rats, which
are normally white. However, internal discoloration was as apparent
in the mice as it was in the rats.
B. Survival
The estimated probabilities of survival for male and female mice
in the control and diarylanilide yellow-treated groups are shown in
Figure 4.
For both male and female mice the Tarone test did not detect a
statistically significant positive association between dosage and
mortality. In the male groups, 74 percent of the high dose, 88
percent of the low dose, and 84 percent of the control mice survived
until the end of the study, despite the sacrifice of five high dose
mice in week 78 and five control mi^e in week 79. In the female
groups 68 percent of the high dose, 86 percent of the low dose, and
26
TIME ON TEST (WEEKS)
FIGURES GROWTH CURVES FOR DIARYLANILIDE YELLOW CHRONIC STUDY MICE
27
TIME ON TEST (WEEKS)
FIGURE 4 SURVIVAL COMPARISONS OF DIARYLANILIDE YELLOW CHRONIC STUDY MICE
28
80 percent of the control group survived until the end of the study,
despite the sacrifice of five high dose mice in week 78 and five
control mice in week 79.
In both sexes survival was adequate for meaningful statistical
analyses of tumor incidence.
C. Pathology
Histopathologic findings on neoplasms in mice are tabulated in
Appendix B (Tables Bl and B2); findings on nonneoplastic lesions are
tabulated in Appendix D (Tables Dl and D2).
There appeared to be no dose- or sex-related increase in the
incidence of neoplasms or toxic changes in the treated versus the
control groups.
With a few exceptions, the same variety of neoplasms occurred
sporadically and at random in the chemically treated and control
groups. No particular organ or system seemed to be the target of
this chemical. Sporadic and unusual problems that occurred in the
treated but not in control animals were as follows: in the integu
mentary system, one mastocytoma affected the subcutaneous tissue of
a high dose female; one squamous-cell carcinoma of the ear affected
a low dose male; and one infiltrating duct carcinoma of the mammary
gland affected one low dose female.
The incidence and variety of nonneoplastic degenerative, pro
liferative, and inflammatory lesions were similar in control and
chemically treated mice.
29
The results of this histopathologic examination did not provide
evidence for the carcinogenicity of diarylanilide yellow in B6C3F1
mice.
D. Statistical Analyses of Results
The results of the statistical analyses of tumor incidence in
mice are summarized in Tables 5 and 6. The analysis for every type
of tumor that was observed in more than 5 percent of any of the
diarylanilide yellow-dosed groups of either sex is included.
None of the statistical tests for mice of either sex indicated a
significant positive association between the administration of diaryl
anilide yellow and an increased tumor incidence in B6C3F1 mice.
To provide additional insight, 95 percent confidence intervals
on the relative risk have been estimated and entered in the tables
based upon the observed tumor incidence rates. In many of the inter
vals shown in Tables 5 and 6, the value one is included; this indi
cates the absence of statistically significant results. It should
also be noted that many of the confidence intervals have an upper
limit greater than one, indicating the theoretical possibility of a
significantly increased rate of tumor incidence induced in mice by
diarylanilide yellow that could not be established under the condi
tions of this test.
30
31
TABLE 5
ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT SPECIFIC SITES IN MALE MICE TREATED WITH DIARYLANILIDE YELLOW3
LjVSW
TOPOGRAPHY: MORPHOLOGY CONTROL DOSE DOSE
Lung: Alveolar/ Bronchlolar Adenoma or Carcinoma 7/47(0.15) 5/49(0.10) 4/49(0.08)
^osed groups received concentrations of 2.5 and 5.0 percent in feed.
^Number of tumor-bearing animals/number of animala examined at site (percent).
cBene«th the Incidence of the control Is the probability level for the Cochran-Armltage test for dose-related trend In proportions when It Is below 0.05; otherwise N.S. - not significant. Departure from linear trend is noted when It is below 0.05 for any comparison. Beneath each dose group incidence is the probability level for the Fisher exact (conditional) test for the comparison of that dose group to the control group when It Is below 0.05, otherwise N.S. - not significant.
''A negative trend (N) Indicates a lower Incidence In a treated group than in a control group.
"Relative risk of the treated group versus the control group Is shown along with the lower and upper limit of the 95* confidence interval for that
relative risk.
TABLE 6
ANALYSES OF THE INCIDENCE OF PRIMARY TUMORS AT SPECIFIC SITES IN FEMALE MICE TREATED WITH DIARYLANILIDE YELLOW*
LOW HIGH TOPOGRAPHY :MOaPHO LOGY CONTROL DOSE DOSE
Lung: Alveolar/Bronchiolar Adenoma or Carcinoma 4/50(0.08) 3/49(0.06) 1/48(0.02) P Values0 'd
Hematopoletic System: Leukemia or Malignant Lymphoma*" 6/50(0.12) 3/50(0.06) 6/50(0.12) P Values0 >(J N.S. N.S. N.S.
% Relative Risk (Control)6 0.500 1.000
Lower Limit 0.085 0.287 Upper Limit 2.200 3.489
Weeks Co First Observed Tumor 97 84 68
Doaed group* received concentrations of 2.5 and 5.0 percent in feed.
Number of tumor-bearing animals/number of animals examined at site (percent).
°Beneath the incidence of the control is the probability level for the Cochran-Armitage test for dose-related trend in proportions when It is below 0.05; otherwise N.S. - not significant. Departure from linear trend is noted when it is below 0.05 for any comparison. Beneath each dose group incidence is the probability level for the Fisher exact (conditional) test for the comparison of that dose group to the control group when it is below 0.05, otherwise N.S. - not significant.
A negative trend (N) Indicates a lower incidence In a treated group than in a control group. 8Relatlve risk of the treated group versus the control group is shown along with the lower and upper limit of the 95% confidence interval for that relative risk.
V. DISCUSSION
Under the conditions of this bioassay, adequate numbers of
chemically treated rats and mice survived for meaningful statistical
analysis of the incidence of late-developing tumors. However, expo
sure to diarylanilide yellow did not result in a positive association
between dietary concentration and increased incidence of any tumor in
either species.
The high concentration administered to both species in the
chronic bioassay was the highest permissible as indicated by the
Guidelines for Carcinogen Bioassay in Small Rodents (Sontag et al.,
1976). These guidelines indicate that a dietary concentration
greater than I percent should not be administered except under
special circumstances (e.g., when the compound is a major component
of the human diet). As human exposure to diarylanilide yellow does
not warrant special exemption, the 5 percent limit applied. Dietary
administration of diarylanilide yellow had no significant effect on
survival or body weight gain in rats or mice of either sex. The only
clinical observation associated with chemical treatment was bright
yellow staining of the fur and mucosal surfaces in both species and
the only sign of toxicity observed during the histopathologic exami
nation was basophilic cytoplasm changes in treated rats.
In rats, no treatment-related increase in the incidence of neo
plasms, nonneaplastic lesions, or toxic effects was evident with the
exception of basophilic changes in hepatocyte cytoplasm in treated
33
males and females. There were, however, two unusual findings: me
tastatic chordoma in 1/49 low dose males, and an osteogenic sarcoma
in 1/49 low dose females.
In mice, no treatment-related increase in the incidence of neo
plasms, nonneoplastic lesions, or toxic effects was evident. There
were, however, three unusual findings: squamous-cell carcinoma of
the ear in 1/49 low dose males, an infiltrating duct carcinoma of the
mammary gland in 1/50 low dose females, and a mastocytoma of the sub
cutaneous tissue in 1/50 high dose females.
The results of this bioassay did not provide evidence for the
carcinogenicity of diatylanilide yellow in Fischer 344 rats or B6C3F1
mice.
34
VI. BIBLIOGRAPHY
Anthony, H.M.. and G.M. Thomas, "Tumors of the Urinary Bladder: An Analysis of the Occupations of 1,030 Patients in Leeds, England." Journal of the National Cancer Institute 45:879-895, 1970.
Armitage, P., Statistical Methods in Medical Research, Chapter 14. J. Wiley & Sons, New York, 1971.
Berenblum, I., editor, Carcinogenicity Testing. International Union Against Cancer, Technical Report Series, Vol. 2. International Union Against Cancer, Geneva, 1969.
Chemical Abstracts Service, The Chemical Abstracts Service (CAS) Ninth Collective Index, Volumes 76-85, 1972-1976. American Chemical Society, Washington, D.C., 1977.
Cox, D.R., Analysis of Binary Data, Chapters 4 and 5. Methuen and Co., Ltd., London, 1970.
Cox, D.R., "Regression Models and Life-Tables." Journal of the Royal Statistical Society, Series "B" 34:187-220, 1972.
Gart, J.J., "The Comparison of Proportion: A Review of Significance Tests, Confidence Limits, and Adjustments for Stratification." International Statistical Institute Review 39:148-169, 1971.
Hawley, G.G., The Condensed Chemical Dictionary, 8th edition. Van Nostirand Reinhold Company, New York, 1971.
Hoover, R. and J. Fraumeni, "Cancer Mortality in U.S. Counties with Chemical. Industries." Environmental Research 9:196-207, 1975.
Kaplan, E.L. , and P. Meier, "Nonparametric Estimation from Incomplete Observations." Journal of the American Statistical Association _53_:457-481, 1958"!
Linhart, M.S., J.A. Cooper, R.L. Martin, N.P. Page, and J.A. Peters, "Carcinogenesis Bioassay Data System." Computers and Biomedical Researcti 7:230-248, 1974.
Miller, R.G., Simultaneous Statistical Inference. McGraw-Hill Book Co., New York, 1966.
Occupational Safety and Health Administration, "Occupational Safety and Health Standards 1910.93c Carcinogens." Federal Register
J38_:974, 1973.
35
Saffiotti, U., R. Montesano, A.R. Sellakumar, F. Cefis, and D.G. Kaufman, "Respiratory Tract Carcinogenesis in Hamsters Induced by Different Numbers of Administration of Benzo (a) Pyrene and Ferric Oxide." Cancer Research 32:1073-1079, 1972.
Society of Dyers and Colourists, Colour Index, Third edition 4^32723305, 1971. ~
Sontag, J.M., N.P. Page, and U. Saffiotti, Guidelines for Carcinogen Bioassay in Small Rodents. Carcinogen Program, Division of Cancer Cause and Prevention, National Cancer Institute, Bethesda, Maryland, NCI-CG-TR-1. DREW Publication No. (NIH) 76-801, February 1976.
Tarone, R.E., "Tests for Trend in Life-Table Analysis." Biometrika 62^679-682, 1975.
U.S. International Trade Commission, Synthetic Organic Chemicals. United States Production and Sales, 1975. USITC Publication 804, U.S. Government Printing Office, Washington, D.C., 1977a.
U.S. International Trade Commission, Imports of Benzenoid Chemicals and Products, 1975. USITC Publication 806, U.S. Government Printing Office, Washington, D.C., 1977b.
Weisburger, E., Chief, Carcinogen and Toxicology Branch, National Cancer Institute, Bethesda, Maryland. Personal communication, December 17, 1976.
Wynder, E.L. , J. Onderflonk, and N. Mantel, "An Epidemiological Investigation of Cancel of the Bladder." Cancer 16:1388-1407, 1963.
36
Review of the Bioassay of Diarylanilide Yellow* for Carcinogenicity
by the Date. Evaluation/Risk Assessment Subgroup of the Clearinghouse on Environmental Carcinogens
September 26, 1977
The Clearinghouse on Environmental Carcinogens was established in May, 1976 under the authority of the National Cancer Act of 1971 (P.L. 92-218). The purpose of the Clearinghouse is to advise on the National Cancer Institute's bioassay program to identify and evaluate chemical carcinogens in the environment to which humans may be exposed. The members of the Clearinghouse have been drawn from academia, industry, organized labor, public interest groups, State health officials, and quasi-public health and research organizations. Members have been selected on the basis of their experience in carcinogenesis or related fields and, collectively, provide expertise in organic chemistry, biochemistry, biostatistics, toxicology, pathology, and epidemiology. Representatives of various Governmental agencies participate as ad hoc members. The Data Evaluation/Risk Assessment Subgroup of the Clearinghouse is charged with the responsibility of providing a peer review of NCI bioassay reports on chemicals studied for carcinogenicity. In this context, below is the edited excerpt from tne minutes of the Subgroup's meeting at which Diarylanilide Yellow was reviewed.
The primary reviewer said that the compound is used as a dye coatiig for yellow lead pencils. It could be a public health concern from the standpoint of people ingesting the dye by chewing on their pencils. Diarylanilide Yellow belongs to the chemical class of diazobenzidines. Some members of this class are reduced by hepatic enzymes to free amines which may be carcinogenic. It was noted that certain bladder carcinogens were not identified until they were tested in appropriate animal models.
The primary reviewer said that the conclusion drawn in the bioassay report was that the study did not provide evidence for the carcinogenicity of Diarylanilide Yellow in either rats or mice. He pointed out, however, that the incidence of pituitary chromophobe adenomas in the treated
* Subsequent to this review, changes may have been made in the bioassay report either as a result of the review or other reasons. Thus, certain comments and criticisms reflected in the review may no longer be appropriate.
37
rats was statistically significant when compared to the controls. A staff pathologist commented that in this particular laboratory, the pituitary tumors were subclassified. If they were considered simply as pituitary adenomas NOS, or had the control pituitary adenomas been sub-classified, they would not have been statistically significant.
The primary reviewer also noted a finding of a single squamous-cell ear carcinoma in a mouse and that this lesion was unreported among the historical control animals. In addition, he commented on a number of other "odd tumors" found in the treated animals. The primary reviewer was critical of the report for not pointing out these tumors in the treated animals since it could mislead readers to believe that there should be no concern about the dye. Another Subgroup member noted a significant increase in the incidence of leukemias and lymphomas in the treated male rats, as well as a decrease in the incidence of hepatocellular carcinomas in the treated male mice. He said that consideration should be given to this phenomenon in evaluating the biological potential of the test compound. Another Subgroup member commented that a survival analysis would be necessary to determine whether there was a true reduction in tumor incidence among the treated animals.
A motion was, made that Diarylanilide Yellow was not carcinogenic under the conditions of test. It was further moved that metabolism studies be done to determine if the compound is reduced to a free amine and if so, consideration be given to a retest in an animal model appropriate for studying bladder carcinogenesis. The motion was seconded and accepted unanimously.
Members present were:
Gerald N. Wogan (Chairman), Massachusetts Institute of Technology
Arnold L. Brown, Mayo Clinic Lawrence Garfinkel, American Cancer Society Joseph H. Highland, Environmental Defense Fund George Roush, Jr., Monsanto Company Sheldon Samuels, Industrial Union Department, AFL-CIO Michael Shimkin, University of California at San Diego Louise Strong, University of Texas Health Sciences Center John Weisburger, American Health Foundation Sidney Wolfe, Health Research Group
38
APPENDIX A
SUMMARY OF THE INCIDENCE OF NEOPLASMS IN RATS TREATED WITH DIARYLANILIDE YELLOW
TABLE Al SUMMARY OF THE INCIDENCE OF NEOPLASMS IN MALE RATS TREATED WITH DIARYLANILIDE YELLOW
. CONTROL(ONTR) LOS DOSE HIGH DOSE 01-0160 01-0195 01-0200
ANIMALS INITIALLY IN STUDY 50 50 50 ANIMALS NECBCPSIED 50 50 50 ANIMALS EXAMINED HISTOFATHOLOGICALLY ** 50 K9 50
* N U M E Z R OF A N I M I . L S KITH TISSUE E X A M I N E D MICROSCOPICALLY * N U M B E R OF A N I M A L S NECROPSIED "EXCLUDES PARTIALLV AUTOLYZED ANIMALS
A-3
TABLE Al (CONTINUED)
CONTROL(ONTB) LOW DOSE HIGH DOSE 01-0160 01-0195 01-0200
*MDITIPLE ORGANS LEUKEMIA, NOS CYELCMONGCfTIC LEUKEMIA
ISPLEEN HYELCMONOCfTIC LEOKFMIA
CIRCULATORY SYSTEM
NONE
DIGESTIVE SYSTEM
iLIVER NEQFLASTIC NODULE HEPATgCELIULAB CARCINOMA
CONTROL (UNTH)C2-0160
501
49** 49
(49)1 12X)
(149)2 («*)
(l»9)
(<*9)1 (2%) 6 (12X)
(U7)
C»8)
1 12S1_
LOB DOSE 02-0195
50
49 49
(49)
(U9)
(1»8)
1 (2X)
(U9)
4 (8%)
(49)
(U9)
t K U K E E E O F A M M A L S HITR T I S S U E E X A M I N E E M I C R O S C O P I C A L L Y * N U M B E R OF A M B A L S N E C F O P S I E D **EXCLUDES PARTIALLY AUTOLYZED ANIMAI.S
*KULTIPLE ORGANS MALIGNANT LYMPHOMA, NOS MALIG.LYMEHOMA, LYMPHOCYTIC TYPE HALIG.LYKEHOMA. HISTIOCYTIC TYPE
*SPLEEN HEMANGIOSARCOMA ANGIOSARCCMA
*BESENTERIC 1. NODE MALIG.LYMEHOMA, HISTIOCYTIC TYPE
tPEYERS PATCH HALIG. LYKEHOHA, HISTIOCYTIC TYPE
CIBCQLATORY SYSTEM
NCNE
CONTROL(DNTR) 05-0160
50
50 ** U9
(50)
(50)
(47) 2 (US) t (9*) 3 (6X)
(50) 1 (2*)
(«9) 1 (2»)
(«0)
(«9)
LOB DOSE HIGH DOSE 05-0195 05-0200
50 50 1
«9 49 49 49
(49) (49) 1 (2X)
(49) (49) 1 (2«)
(«9) (49)
1 (2%) 1 (2%) 4 (8X) 3 <6X)
(49) (49)
1 (2%) 1 (2%) 1 (2%) 1 (2%)
(4b) (47) 1 (2»)
1 (2*) 1 (2%)
(35) (43) 1 (3X)
(48) (49) 1 (2%)
* N U M B E R O F A N I M A L S WITH TISSUE E X A M I N E D f l lCBOSCOPICALLY * K U K B E R OF A M P U L S N E C R O P S I E D **EXCLUDES PARTI UiY AUTOLYZED ANIMALS
*MESENTERIC L. NODE (aoj (36) (45) BiiISSiNI_IXi3PfloaAJ._!HXEp_TiE£ 1_L2SI_
* N U M B E R OF A N I M A L S BITH TISSUE E X A M I N E D MICROSCOPICALLY * N U M B E R OF A N I M A L S NECROPSIED "EXCLUDES PARTIALLY AUTOLYZID ANIMALS
B-6
TABLE B2 (CONTINUED)
CONTROL (ONTR)06-0160
L0¥ DOSE 06-0195
HIGH DOSE 06-0200
IEFYERS PATCUMALIGNANT LY HPHOHA, NOS
tJIJDHOKMAHG. LYMPHO3A, HISTIOCYTIC TYPE
<«9) 1 (2*)
(«9)
<«6)
(«6) 1 (2%)
(l»7)
(U7)
CIHCDLATCKY SYSTEM
NCNE
DIGESTIVE SYSTEM
tLIVERHEPATOCELLOL\B CARCINOMA
(149) 2 («%)
(«7) (U6)
ORINABY SYSTEM
NONE
ENDOCRINE SYSTEM
*PITUITARYADENOMA, NOSCHROKOPHOBE ADENOMA
• ADRENALEHEOCHROMOCYIOMA
(U2)
(a7)
(39) 1 (3X)
(1»6) 1 (2X)
(l*U)
1 (2«)
(US)
REPBCDOCTIVE SYSTEM
*BAMMARY GLANDINFILTRATING DUCT CARCINOMA
IOTEROSENDOBETRIAL STROMAL SARCOMA
(50)
(«9)
(50) 1 (2%)
(17) 1 (2«)
(50)
(U6)
NEEVOOS SYSTEM
NCNE
SPECIAL SENSE ORGANS
NONE
t N U M B E R* N H M B E E
OF OF
A N I M U L S A N I M A L S
HITH TISSUE NECROPSIED
E X A M I N E D
.
MICROSCOPICALLY
_ _ . .
B-7
TABLE B2 (CONCLUDED)
*CONTROL (UNT 06-0160
B) LOU DOS306-0195
HIGH DOSE 06-0200
BUSCDLOSKEiriAL SYSTEH
NONE
BODY CAVITIES
NONE
ALL CTHEP SYSTEMS
NONE
ANIMAL EISEOSITICN SUMHAEY
ANIKALS INITIALLY IN STUDY NATUFAL EEATHd MORIBUND SACRIFICE SCHECULEE SACRIFICE ACCIDENTALLY KILLED TERMINAL SACBIFICE ANIMAL MISSING
50 3 2 5
HO
5061
U3
50 8 3 5
31*
3 INCLUDES AUTCLYZED ANIMALS
TUMOR SUMMARY
TOTAL ANIMALS KITH PRII1ABY TUMCBS* TOTAL PEIMABY TUMOBS
12 15
1010 9
9
TOTAL ANIMALS WITH BENIGN TUMORS TOTAL BENIGN TDMORS
1 1
33 2
2
TOTAL ANIMALS WITH MALIGNANT TUMORS TOTAL MALIGNANT TUMORS
11 1H
77 6
6
TOTAL ANIMALS HITH SEC3NDABY TUKORS* TOTAL SECONDARY TDHOBS
1 1
TOTAL ANIMALS SITH TUM3HS UNCERTAINBENIGN OR MALIGNANT
TOTAL UNCERTAIN TUMORS 1 1
TOTAL ANIMALS KITH TUMORS UNCERTAINFFIMABY CE MITASTATIC
TOTAL UNCERTAIN TUMORS
* PRIMARY TUMORS: ALL TUMORS EXCEPT SECONDARY TUHORS # SECONDARY TUMORS: METASTATIC TUMORS OR TUHORS ISVASIVE INTO AN ADJACENT ORGAN
B-8
APPENDIX C
SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN RATS TREATED WITH DIARYLANILIDE YELLOW
TABLE Cl SUMMARY OF THE INCIDENCE OF NONNEOPLASTIC LESIONS IN MALE RATS
TREATED WITH DIARYLANILIDE YELLOW
* N O M E E R OF A N I M A L S W I T H TISSOE E X A M I N E D MICROSCOPICALLY * N U M B E R OF A N I M A L S N E C R O P S I E D **EXCLUDES PARTIALLY AUTOLYZED AKIMALS
C-3
TABLECl (CONTINUED)
... ... CONTHOL(ONTE) LOW DOSE 01-0160 01-0195
tHESENTERIC L. NODE (149) (t6) HYPESPLASIA, PLASHA CELL 1 (2%)
t N U M B E R OF A N I M A L S HITH TISSUE E X A M I N E D MICROSCOPICALLY * N U M B E R OF A N I M A L S NECFOPSIED **EXCLUDES PARTIALLY AJTOLYZED ANIMALS
•MEDIASTINAL L. NODE (UO) (36) HYPERPLASIA, NOS 1 (3%)
tLUMBAR LYRPH NODE (<«0) (36) HYPERPLASIA, NOS 1 (3%)
•HESENTERIC L. NODE («0) (36) __HI£EEEIiSIA^_JJ2£
t S O M B E R OF A N I F A L S SITH TISSUE E X A M I N E D MICROSCOPICALLY * N O H E E K OF A N I t A L S N E C R O P S I E D **EXCHJDES PARTIALLY AUTOLYZED ANIMALS
CONTROL (UNTB) LOW DOSE HIGH DOSE 06-0160 06-0195 06-0200
1 (2X) 1 (2*) 1 <2«)
(49) (<»6) (47) 1 (2%)
(50) C»D (45) 1 (2%)
(49) («9) (47) 4 (8%)
1 (2X) 1 (2%)
2 («%) 1 (2%) 1 (2S)
(50) (44) (45) 1 (2%)
1 (2%)
(50) (4U) (45) 1 <2X)
16 (32*) 1 <2S)
(50) («4) (45) 1 (2S)
(t2) 139) (44) 2 (5*) 1 <3J)
("»D (41) (44) 1 (2S)
1 (2X> 2 (5%) 1_JL2SI_ . l-l&L
.——-.— ——«———
* NUMBER 0" ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY » NUMBER OF ANIMALS NECROPSIED
D-9
TABLE D2 (CONTINUED)
111 " •"«"—™—g»™-»-""»~i"> i-mr-- Jr _ JT -J.IT _ •••••M~HWMrjH«-«
C O N T B O L ( D N T H ) LOU DOSE 06-0160 06-0195
REPRODUCTIVE SYSTEM
tOTERDS (<»9) (47)H Y D R O B E T R A 5 (10») ^ (15%) ABSCESS, NOS 1 (2%) NECHOJIS , FAT 1 (2*) CALCIMCATION, NOS 1 <2S)
* UTERUS/ KNDOMETRIOH (U9) (47) INFLAMMATION. SUPPURATIVE 2 («%) INFLAMMATION, ACUTE 1 (2%) H Y P E R l ' L A S I A , NOS 1 (2S) H I P E f c l ' L A S I A , CYSTIC 32 (65%) 34 (72%)
«OVARY/07IDUCT <«9) (47) INFLA1HATION, NOS ABSCE3S. NOS 1 (2%) INFLASMATION, CHRONIC
I O V A B Y / P A B O V A R I A N (49) (47) STEATITIS ABSCESS, NOS 2 ( 4 % ) INFLAHHATION, ACUTE/CHRONIC 1 (2%)
*OVARY («8) (42) CYST, NOS 6 (13X) 7 (17%) I N F L A M M A T I O N . N O S I N F L A M M A T I O N , S U P P U B A T I V E 1 (2%) ABSCISS, NOS I N F L A M M A T I O N . ACUTE/CHRONIC 2 (5%) I N F L A M M A T I O N , CHRONIC 1 (2%) HYPE1IPLASIA, EPITHELIAL 1 (2*)
HEHVCUS l iYSTEW
« E R A I N / K E N I N G E S (49) ( 4 6 ) LYMP!10CYT1C I N F I L T R A T E
I E R A I N (49) (46) H Y B R D C E P H A L U S , NOS 1 (2X)
SPECIAL SENSE O R G A N S
* E Y E / L A C R I H A L G L A N D (50) (50) iJlPFRPLA£Ii4._JjaS_ 1 J2J1_
t NOHEEB OF ANIMALS WITH TISSUE EXAMINED MICBOSCOPICALLY * NUMBEF OF ANIMALS NECROPSIED
.
HIGH DOSE 06-0200
(46) 7 (15%)
(46)
3 (7%) 1 (2%)
24 (S2%)
(46) 1 (2%) 1 (2%) 1 (2%)
(»6) 1 (2%)
(46) 7 (15%) 2 (4%)
1 (2%)
(47) 2 (4%)
(47)
(50)
D-IO
TABLE D2 (CONCLUDED)
CONTROL (UNTB)Ofa-0160
LOH DOSE 06-019S
HIGH DOST jb-0200
HUSCULOSKELETAL SYSTEM
DOME
BOEY CAVITIES
•MEDIASTINUMINFLAMMATION, ACDTENECROSIS, NO:;
•PERITONEUMINFLAMMATION, NOSABSCESS, NOS
(50)
(50)
(50) 1 (2%)
i (2*)
(50)
(50)
(50) 1 (2X) 1 (2%)
ALL OTHEK SYSTEM
•MULTIPLE ORGANSAMYLOIDOSIS
CMENTUK PIRIVASCULITJS
(50) 1 <2X)
1
(50) (50)
SPECIAL MORPHOLOGY SUMMARY
NO LFSION REIOBTED 2 AUTO/NECROPSY/HISTO PERF 2 AOTO/NECBOPSY/NO HISTO
t NOHEER OF ANIMALS WITH TISSUE EXAMINED MICROSCOPICALLY * NUMBER OF ANIMALS NECROPSIED